A. Field the Invention
This invention relates to chemically modified allergens and to the process for the preparation thereof. More particularly, this invention relates to a new type of chemically modified allergens, which are effective for treating allergic diseases, as well as to the method for the preparation thereof.
Description of the Related Art
It is already known that many people suffer from troubles of allergic type; the most common symptoms are asthma, hay fever and conjunctivitis, urticaria. The mechanism that causes such troubles is usually due to an allergic sensitization state consisting in the hyperproduction of antibodies of the IgE class which have an affinity for ubiquitous allergens, as for instance, pollens, house dust mites, fungi spores, and so on. The antibodies of the IgE class do not exert any protective role usually exerted by the antibodies of the other classes, but in addition they cause a complex cellular reaction when they react with allergens for which they have an affinity, as they have the capability of binding to tile membrane of the mast cells of the mucosa and to the basophilic leucocytes, said reaction resulting in the release of vasoactive amines (for instance histamine) and other compounds which are the actual mediators of the allergic reactions and are responsible for the same.
In order to reduce or to remove the allergic troubles, the specific hyposensitizing therapy has been practised for many decades. It consists in administering to the allergic patient, generally through the subcutaneous injection route, progressively increasing doses of those allergens to which the patient is sensitive, such allergens being identified during previous diagnostic investigation. The traditional allergenic extracts are made up of water solutions of allergens having a pH value close to neutrality (pH 7.0-7.4) and an osmotic strength similar to that of the serum.
The mechanisms through which said specific hyposensitizing therapeutic treatment results in the decrease or in the disappearance of the allergic symptoms are manifold; the most known and certainly the most important of them is the following: the repeated injection of the allergen induces in the organism the production of allergen-specific antibodies of the IgG class, called "blocking antibodies", capable of reacting with the allergen and of preventing the same from reacting with the IgEs so causing the pathogenic reaction previously outlined.
In order that said specific hyposensitizing therapeutical treatment could be efficient, it is necessary that the dose of the allergen which is injected in total is high.
Though it is universally accepted because of its effectiveness, said specific hyposensitizing therapeutic treatment shows however some drawbacks which are due to the unwanted reactions that can occur following the injection of the allergen. Indeed, local reactions can occur just at the injection site (large red patchs, reddening, itch, and so on), or systemic reactions (rhinitis or asthma, and anaphylactic shock).
In order to reduce such undesired reactions and hence to make the specific hyposensitizing therapy safer, allergenic extracts of a depot type, i.e. slow-absorption allergenic extracts, have been realized in the most recent years, in which extracts of the allergens are precipitated or adsorbed with aluminum hydroxide, tyrosine or calcium phosphate, in order to slow down the absorption in the organism and then to reduce the reactivity: with such type of extracts, patients can better tolerate the inoculation of the extract and with a less number of undesired reactions.
Though such depot type allergenic extracts represent an improvement, however they are not the optimal and definitive solution to the problem because a certain percentage of patients responds to them also with undesired reactions, both local and systemic. In an attempt to obviate such drawbacks, chemical modifications of the allergens have been tried, such as decreasing their allergenic reactivity substantially (i.e., the capability of reacting with the IgEs bonded to the tissue mast cells and then of causing the pathogenic reaction), while keeping their immunogenic power unchanged (i.e., the capability of inducing the formation of blocking antibodies of the IgG class). Some authors have employed the term "allergoids" to point out the allergens so modified.
A method proposed to produce modified allergens having in part the required features is the denaturation method with 8 M urea. Indeed, it has been shown that, by exposure of the antigen E of Ambrosia elatior pollen (a very important allergenic plant growing in the United States) to the dissociating action of an 8 M solution of urea, such antigen lost a large amount of its capability of reacting with the IgEs, though it kept the hyposensitizing capability after injection for therapeutical purposes. However, tests in human subjects, following to such quite encouraging experiments performed on mice, gave conflicting results. Moreover, the method disclosed above seems to be employable just for the antigen E of Ambrosia elatior, made up of two polypeptidic subunits put together by non-covalent bonds, which is particularly sensitive to the denaturing action of dissociating agents, such as 8 M urea.
Another method which is already known for obtaining the chemical modification of allergens, is the treatment with aldehydes, the most employed of them being formaldehyde or glutaraldehyde. The reactions between aldehyde functions and protein amino groups are described for instance by R. E. Feehey, G. Blankenhorn and H. B. F. Dixon, Adv. Prot. Chem. 29, 135-203, 1975; it is to be kept in mind that almost all allergens are chemically constituted of glycoprotein molecules, in which the protein component is the part relevant to give its features of immunologic specificity.
It has been shown that by treating with formaldehyde Lolium perenne's and Ambrosia's allergens, a 100-10,000 times decrease in the allergenic reactivity is obtained with respect to the native allergen. The conditions for the chemical modification are described by Marsh in "The Antigens" vol. III, pg. 317, Ed. Sela M., Academic Press, New York. The product so obtained keeps a satisfying immunogenic capability when injected for therapeutic purposes, but the reactivity changes from patient to patient in a wide degree: as a consequence, doses as well as dosage schemes which may be optimal for some patients, might happen to be not so for others.
The reaction with glutaraldehyde, as this is a bifunctional aidehyde, causes the formation of a mixture of polymers having various molecular weights (see for instance R. Patterson, J. Immunol. 110, 1413, 1973, in which the antigen E of Ambrosia is subjected to polymerization with glutaraldehyde, so obtaining a product having an allergenic activity reduced by 10-100 times, or otherwise see D. M. Moran and A. W. Wheeler, Int. Archs. Allergy and Applied Immunology, 50, 693, 1976, in which a similar procedure is applied to allergens of Phleum pratense). However, the polymerization method employing glutaraldehyde shows two drawbacks:
1) the removal of macromolecules from the organism, such as those which are obtained by means of that procedure (from 2.times.10.sup.5 to 2.times.10.sup.7 daltons) is difficult, so that toxicity is likely to be high;
2) the final addition of glycine, lysine or other amino acids or compounds containing at least one amino group for stopping the reaction results in products of synthesis between glycine and glutaraldehyde, which products have no relationship with the native allergen and are likely to bring about new allergenic determinants which are useless in regard to the therapeutic effects.
Another chemical procedure for modifying allergens is the object of the UK patent No. 1282163, in which the reaction process of Gramineae pollens allergens with an inorganic cyanate, or with a polyaldehyde or carbodiimide is described. The object of the process consists in the preparation of modified allergenic material, substantially insoluble or just partially soluble in water.
The alkaline cyanate procedure is carried out in an acid medium, in particular at a pH of 5. The occurring reaction is the condensation of the amino groups of the allergen with the carboxyl groups (activated by cyanic acid in the form of a mixed anhydride) of the allergen itself (Methods in Enzymology 25, 579, 1972). Any way, by means of the procedure disclosed in the UK patent No. 1,282,163, the allergenic material is polymerized till it becomes insoluble in water or in water solutions.
The procedures of the prior art described above result anyway in all cases in the formation of polymeric derivatives of the allergen, so that they are characterized by a lower bond valence with respect to the specific antibodies of the IgE class.